Journal: bioRxiv

Article Title: Overcoming Immunological Resistance Enhances the Efficacy of a Novel anti-tMUC1 CAR T Cell Treatment Against Pancreatic Ductal Adenocarcinoma

doi: 10.1101/642934

Figure Lengend Snippet: A . The architecture of three different CAR constructs used in this study. In the original construct (tMUC1-CAR), scFv of TAB004 antibody is linked to CD28 transmembrane (TM) domain followed by CD28 and CD3ζ intracellular domains in a retroviral plasmid. CD8a leader sequence was used as signal peptide for cell membrane expression of the CAR. In the CTL-CAR construct, scFv of TAB is removed. In the CAR-mKate construct, mKate2 gene was fused to the C-terminus of CAR flanking with a GA linker. B. CTL-CAR and tMUC1-CAR expression were measured by flowcytometry using FITC-conjugated anti-myc tag antibody, and expression level in CD4+ and CD8+ primary T cells on day 12 after infection are shown. On average 42% of T cells expressed tMUC1-CAR. C . Bright field (top left) and fluorescent image (top right) of live T cells expressing CAR-mKate plated in 35mm poly-D-lysine coated MatTek dish imaged by DeltaVision workstation (Applied Precision). Projection image of a T cell expressing CAR-mKate (bottom left), and one Z image of the CAR-mKate T cell (bottom right) illustrating the ring-like structure around the cells formed by CAR-mkate expression, which indicates even distribution of CAR molecules on the T cell membrane. D. Light and fluorescent image of CAR-mKate T cells binding to MUC1 expressing cancer cell (HPAFII). HPAFII cells were incubated with CAR-mKate T cells for 4 hours, then T cell were remove, HPAFII cell was washed and imaged using DeltaVision microscope. The intense red signal observed between CAR T cell and HPAFII indicates co-localization and strong binding of CAR molecules which suggests forming an immunological synapse. Nuclei are stained with Hoechst nuclei blue dye in C and D.

Article Snippet: The CAR expression level was quantified using myc tag-FITC staining (Cell Signaling Technology, Danvers, MA).

Techniques: Construct, Plasmid Preparation, Sequencing, Expressing, Infection, Binding Assay, Incubation, Microscopy, Staining

Journal: Journal of Microbiology (Seoul, Korea)

Article Title: Genetically Engineered CLDN18.2 CAR-T Cells Expressing Synthetic PD1/CD28 Fusion Receptors Produced Using a Lentiviral Vector

doi: 10.1007/s12275-024-00133-0

Figure Lengend Snippet: The characteristics of CLDN18.2 CAR-T cells. A Expression of CAR on T cells. Activated T cells were transduced using lentivirus encoding synthetic CLDN18.2-CAR constructs, and CAR expression level was assessed using flow cytometry after staining with FITC-labeled goat anti-mouse IgG F(ab’)2. B Proliferation curves of CAR-T cells and non-transduced T cells. In all cases, the cells expanded 200-fold 11 days post-transduction

Article Snippet: CAR expression levels were examined by staining with goat anti-mouse IgG F(ab’)2 (Jackson ImmunoResearch) using flow cytometry at 4, 6, 8, and 11 days after transduction.

Techniques: Expressing, Construct, Flow Cytometry, Staining, Labeling, Transduction